This application is a 371 national stage application of PCT/AU95/00670, filed Oct. 12, 1995.
This invention relates to saponin preparations, particularly to saponin preparations based on defined compositions of purified saponin fractions derived from the bark of Quillaja saponaria Molina. The invention also extends to immunostimulating complex (iscom) matrices prepared using these saponin preparations, as well as to immunogenic iscoms in which immunogens are incorporated into or associated with an iscom matrix. Such immunogens will usually be proteins or peptides derived from bacteria, viruses or other microorganisms, but they may, in addition, be any other protein, peptide or other chemical entity which can induce an immune response.
The saponin preparations of this invention, and iscom matrices prepared using them, have particular activity as adjuvants, that is as products which result in a specific increase in the immunogenicity of a vaccine component
The adjuvant properties of saponin have been long known as has its ability to increase antibody titres to immunogens. As used herein the term xe2x80x9csaponinxe2x80x9d refers to a group of surface-active glycosides of plant origin composed of a hydrophilic region (usually several sugar chains) in association with a hydrophobic region of either steroid or triterpenoid structure. Although saponin is available from a number of diverse sources, saponins with useful adjuvant activity have been derived from the South American tree Quillaja saponaria Molina. Saponin from this source was used to isolate a xe2x80x9chomogeneousxe2x80x9d fraction denoted xe2x80x9cQuil Axe2x80x9d (Dalsgaard, 1974).
Acute toxicity is a major concern for both the veterinary and human use of Quil A in vaccine preparations. One way to avoid the acute toxicity of Quil A is the use of iscoms, an abbreviation for Immuno Stimulating COMplexes. This is primarily because Quil A is less toxic when incorporated into iscoms, because its association with cholesterol in the iscom reduces its affinity for cholesterol in cell membranes and hence its cell lytic effects. In addition, a lesser amount of Quil A is required to generate a similar level of adjuvant effect. Iscoms are small, cage-like structures generally 30 to 40 nm in diameter which retain this structure on freeze drying. The final formulation of a typical immunogenic iscom with an optimal amount of immunogenic protein is a weight ratio of Quil A, cholesterol, phosphatidyl choline, and protein (1:1:1:5). Such a typical iscom is estimated to contain 5 to 10% by weight Quil A, 1 to 5% cholesterol and phospholipids, and the remainder protein. Peptides can be incorporated into iscoms either directly or by chemical coupling to a carrier protein (e.g. influenza envelope protein) after incorporation of the carrier protein into iscoms.
As an adjuvant, the iscom confers many advantages including powerful immunostimulatory effects, low toxicity, ability to induce both cellular (including CTL) and humoral responses, and it is inexpensive in both reagent and manufacturing cost. However, in the past, iscoms have had two major disadvantages; firstly, the Quil A used in their preparation was a complex and ill defined mixture of a biologically-derived product, and batch-to-batch variation was therefore to be expected; and secondly, iscoms still showed injection-site reactivity and low but detectable in vivo toxicity.
Since the recognition of the adjuvant activity of Quil A (Dalsgaard, 1974) several groups have further fractionated this material into a number of xe2x80x9cpurifiedxe2x80x9dcomponents (Morein et al., Australian Patent Specification No. 632067; Kersten, 1990; Kensil, 1988; Kensil 1991). These components were subsequently shown to have variable properties especially in regards to adjuvant activity, haemolytic activity and ability to form iscoms. The use of purified Quil A components conferred two potential advantages for their use in a human vaccine. Firstly, the purified component could be characterized and therefore made reproducibly; and secondly, the components could be selected for optimal usefulness.
The immunomodulatory properties of the Quil A saponins and the additional benefits to be derived from these saponins when they are incorporated into an iscom have been described in various publications, e.g. Cox and Coulter, 1992; Dalsgaard, 1974; Morein et al., Australian Patent Specifications Nos. 558258, 589915, 590904 and 632067. In Australian Patent Specification No. 632067, the separation of a preparation of Quil A into three distinct fractions called B4B, B3 and B2 is described, along with HPLC chromatographic procedures for this fractionation. More carefully defined and controllable procedures for the fractionation of Quil A have now been devised which result in three major fractions with increasing degrees of hydrophobicity in the purification system used.
In work leading to the present invention, it has now been shown that saponins derived from Quillaja saponaria can be separated into fractions with differing chemical and biological properties, including the important biological properties of adjuvant activity, haemolytic activity, ability to form iscoms and in vivo toxicity, and that particular compositions of these fractions can be prepared to form novel saponin preparations which are capable of forming good iscoms, having optimal adjuvant activity but minimal haemolytic and toxic activity.
According to the present invention, there is provided a saponin preparation comprising saponins of Quillaja saponaria, said preparation comprising from 50 to 90% by weight of Fraction A of Quil A (as herein defined) and from 50% to 10% by weight of Fraction C of Quil A (as herein defined).
Preferably, the saponin preparation comprises from 50% to 70% by weight of Fraction A and from 50% to 30% by weight of Fraction C. A particularly preferred preparation comprises about 70% by weight of Fraction A and about 30% by weight of Fraction C.
The term xe2x80x9cQuil Axe2x80x9d is used throughout this specification and in the claims as a generic description of a semi-purified saponin fraction of Quillaja saponaria. 
The saponin preparation may, if desired, include minor amounts (for example up to 40% by weight) of other adjuvant materials with desired immunomodulatory properties, including minor amounts of Fraction B of Quil A or of other saponins. Examples of other saponins or other adjuvant materials which are suitable for inclusion in this preparation are described in Australian Patent Specification No. 632067, incorporated herein by reference.
As described above, it is known that in order to prepare an immunostimulating complex (iscom) matrix, Quil A, a sterol such as cholesterol and optionally a lipid such as phosphatidyl choline, must be included in the reaction mixture.
In accordance with another aspect of the present invention there is provided an immunostimulating complex (iscom) matrix comprising a saponin preparation, a sterol and optionally a lipid, wherein the saponin preparation comprises from 50 to 90% by weight of Fraction A of Quil A (as herein defined) and from 50% to 10% by weight of Fraction C of Quil A (as herein defined).
Preferably, in such an iscom matrix the sterol is cholesterol, and the lipid (which is optionally present) is a phospholipid such as phosphatidyl choline.
In yet another aspect, this invention provides an immunogenic iscom which comprises an iscom matrix as described above having at least one immunogen incorporated into or associated with the iscom matrix.
An iscom matrix or an immunogenic iscom in accordance with the present invention may be prepared by techniques which are well known to persons skilled in the art, and which are described in detail in the publications Cox and Coulter, 1992 and Morein et al., Australian Patent Specifications Nos. 558258, 589915, 590904 and 632067, the disclosures of which are incorporated herein by reference.
The immunogen which is incorporated into or associated with the iscom matrix in accordance with this invention may be any chemical entity which can induce an immune response in an individual such as (but not limited to) a human or other animal, including but not limited to a humoral and/or cell-mediated immune response to bacteria, viruses or other microorganisms.
The specific immunogen can be a protein or peptide, a polysaccharide, a lipopolysaccharide or a lipopeptide; or it can be a combination of any of these. Particularly, the specific immunogen can include a native protein or protein fragment, or a synthetic protein or protein fragment or peptide; it can include glycoprotein, glycopeptide, lipoprotein, lipopeptide, nucleoprotein, nucleopeptide; it can include a peptide-peptide conjugate; it can include a recombinant nucleic acid expression product. Examples of such immunogens include, but are not limited to, those that are capable of eliciting an immune response against viral or bacterial hepatitis, influenza, diphtheria, tetanus, pertussis, measles, mumps, rubella, polio, pneumococcus, herpes, respiratory syncytial virus, haemophilias influenza, chlamydia, varicella-zoster virus, rabies or human immunodeficiency virus.
The present invention also extends to a vaccine composition comprising as the active component thereof either (i) an immunogenic iscom as broadly described above or (ii) an iscom matrix as broadly described above and at least one immunogen, together with one or more pharmaceutically acceptable carriers and/or diluents.
The formulation of such vaccine compositions is well known to persons skilled in this field. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example, in Remington""s Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
It is especially advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the human subjects to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and/or diluent. The specifications for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active ingredient for the particular treatment.
In yet another aspect, the present invention extends to a method of eliciting or inducing an immune response in an individual, which comprises administering to the individual an immunologically effective amount of a vaccine composition as broadly described above.
As previously mentioned, the individual may be a human or other animal, including a livestock animal (eg. sheep, cow or horse), laboratory test animal (eg. mouse, rat, rabbit or guinea pig), companion animal (eg. dog or cat) or wild animal.
An immunologically effective amount means that amount necessary at least partly to attain the desired immune response, or to delay the onset of, inhibit the progression of, or halt altogether, the onset or progression of the particular condition being treated. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the capacity of the individual""s immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
The purification of crude aqueous Quil A extract to Fractions A, B and C of Quil A is described in detail in Example 1 hereinafter. It should be understood that this purification procedure is included by way of example only, and that fractions functionally similar or equivalent to Fractions A, B and C can be prepared by diverse other chromatographic procedures.
For the purposes of identification of Fractions A, B and C referred to herein, reference may be made to the purification procedure of Example 1. In general terms, in this procedure Fractions A, B and C are prepared from the lipophilic fraction obtained on chromatographic separation of the crude aqueous Quil A extract and elution with 70% acetonitrile in water to recover the lipophilic fraction. This lipophilic fraction is then separated by semipreparative HPLC with elution using a gradient of from 25% to 60% acetonitrile in acidic water. The fraction referred to herein as xe2x80x9cFraction Axe2x80x9d or xe2x80x9cQH-Axe2x80x9d is, or corresponds to, the fraction which is eluted at approximately 39% acetonitrile. The fraction referred to herein as xe2x80x9cFraction Bxe2x80x9d or xe2x80x9cQH-Bxe2x80x9d is, or corresponds to, the fraction which is eluted at approximately 47% acetonitrile. The fraction referred to herein as xe2x80x9cFraction Cxe2x80x9d or xe2x80x9cQH-Cxe2x80x9d is, or corresponds to, the fraction which is eluted at approximately 49% acetonitrile.
When prepared as described herein, Fractions A, B and C of Quil A each represent groups or families of chemically closely-related molecules with definable properties. The chromatographic conditions under which they are obtained are such that the batch-to-batch reproducibility in terms of elution profile and biological activity is highly consistent.
Fractions A, B and C as described above have been studied for their adjuvant activity, haemolytic activity and ability to form iscoms, and the results are summarized in Table 1:
Surprisingly, it has now been found that particular combinations of Fractions A and C, more particularly combinations of from 50 to 90% by weight of Fraction A with from 50 to 10% by weight of Fraction C (with 0% of Fraction B), result in a saponin preparation which has the desirable properties of A (good 5 iscom formation and low haemolytic activity) and the benefits of C (good adjuvant activity). In one particularly preferred saponin preparation of this invention, the ratio of 7 parts A: 0 parts B: 3 parts C (=7,0,3; or QH703) has been found to provide very good adjuvant activity, to form iscoms easily yet to have a much lower haemolytic activity than would be expected from the component fractions. It is to be understood, however, that the present invention extends to other saponin preparations ranging from 5 parts A: 0 parts B; 5 parts C (=5,0,5; or QH505) to 9 parts A: 0 parts B; 1 part C (=9,0,1; or QH901).
The following Examples describe a method for the purification of A, B, and C; and compare pure A (10,0,0), pure B (0,10,0), pure C (0,0,10) and the mixture QH703 (7,0,3) in terms of adjuvant activity, haemolytic activity, ease of iscom formation and induction of IL-1, a marker for immunomodulatory activity. Data is also included to demonstrate the pre-clinical safety profile of the saponin preparation of this invention and iscom matrix made therefrom, as well as the clinical safety of this iscom matrix. The overall conclusions from this data is that a mixture of A and C, roughly in the ratio 7:3 (=7,0,3; or QH703) is an optimal ratio of purified saponins from which to form iscom matrix or immunogenic iscoms.
Further features of the present invention are more fully described in the following Example(s). It is to be understood, however, that this detailed description is included solely for the purposes of exemplifying the present invention, and should not be understood in any way as a restriction on the broad description of the invention as set out above.